The present invention relates to a method of measuring changes in optical properties of layered materials, including using a sensor employing a propagating surface mode or waveguide mode in the layered materials for use in biological, biochemical and chemical testing. More particularly the present invention relates to an immunosensor for monitoring the physical interactions between two biologically relevant molecules.
A surface plasmon resonance is the oscillation of free electrons that exists at a metal boundary induced by a time varying electric field. The phenomenon of surface plasmon resonance (SPR) can be used to detect minute changes in the refractive index of layers near a metal surface. The SPR is affected by the refractive index of the material adjacent to a metal surface and it is this dependence that forms the basis of the sensor mechanism.
In the case of immunosensors, as a reaction between an antigen and an antibody proceeds, the surface properties change from their original state. While antibodies are immobilized on the surface, the properties of the surface change when a solution containing a corresponding antigen is brought into contact with the surface. This interaction allows the antigen to bind with the antibody. The change in the optical properties of the surface can then be monitored with a suitable sensor.
Generally, SPR may be achieved by using the evanescent wave that is generated when a P-polarized light beam is totally internally reflected at the interface between two media, e.g. the interface between a liquid medium such as a blood serum, which has a positive dielectric constant, and a metallic medium, such as a metal film, which has a negative dielectric constant. The SPR is excited when the incident angle of the light beam is tuned to a particular angle of incidence at which the electrons in the metallic medium resonantly absorb the energy of the light beam. As a direct consequence the energy in the reflected light beam is strongly diminished. This process is referred to as attenuated total reflection (ATR).
SPR biosensors work on the principle of measuring the change in the ATR when the optical properties of the medium adjacent to the metal are changed. Specifically, antigens in a first solution are initially caused to be immobilized on the metal surface. The optical properties of the surface change when a second solution, sometimes referred to as the analyte medium, which contains a corresponding antibody, is brought into contact with the surface thus allowing the antigen to bind with the antibodies. The angular sensitivity of the ATR feature to adsorbed biological layers on the metal surface has made the SPR device a suitable sensor to monitor the binding reaction.
One particular form of SPR, commonly referred to as the Kretschmann mode, is more properly described as a single boundary mode since the surface wave achieves its peak value at only a single metal/dielectric interface. In practice a sensor system makes use of a light beam that is incident to the surface and reflected. Commonly this is a divergent beam from a light emitting diode (LED) followed by a lens system to produce a slightly focused incident beam with an angular spread of a few degrees. The reflected light beam is generally captured in an optical detector, such as in the form of a linear pixel array. The design of many sensor systems is based on the detection of the angular shift of the entire ATR pattern due to the antibody/antigen binding reaction. A paper published under the title, xe2x80x9cSurface plasmon resonance for gas detection and biosensingxe2x80x9d, by Lieberg, Nylander and Lundstrom in Sensors and Actuators, Vol. 4 at page 299, further describes the SPR technique recited above, the entire paper is hereby incorporated herein by reference.
A more sensitive surface plasmon sensor is described in U.S. Pat. No. 5,846,843 to Simon, which discloses a long range SPR sensor, the disclosure of which is incorporated herein by reference. The sensor includes a first dielectric medium and a second dielectric medium having approximately matching indices of refraction. A thin metal film is located between the first and second dielectric media. A beam of electromagnetic radiation is introduced into the sensor layer assembly in a manner that causes the long-range surface plasmon resonance to occur. This resonance may be achieved through the formation of diffraction gratings at each of the metal/dielectric interfaces as described in U.S. Pat. No. 5,846,843. The long-range surface plasmon (LRSP) can also be termed a double boundary mode as the surface wave achieves its peak value at both metal/dielectric interfaces. The basic diffraction-coupled LRSP phenomenon is further described in a paper entitled, xe2x80x9cAttenuated Total Reflectance From a Layered Silver Grating with Coupled Surface Wavesxe2x80x9d, by Zhan Chen and H. J. Simon in the Journal of the Optical Society B 5, 1936 (1988), the entire paper is incorporated herein by reference.
The method of detecting the binding reaction between the antibody and antigen is identical for both the single boundary Kretschmann mode and the double boundary LRSP mode. In both cases the multilayer assembly is rotated around an axis perpendicular to the plane of incidence to produce a scan in the incident angle. There exists an optimum angle, sometimes referred to simply as the plasmon angle, at which the phenomenon of surface plasmons is observed by manifesting itself as a sharp attenuation of the total reflected (ATR) beam. This angle sensitively depends upon the index of refraction of the second dielectric medium, which is commonly a liquid serum. Thus, the binding reaction between the antibody and antigen changes the value of the plasmon angle. The angle of incidence of the beam is varied to detect the plasmon angle. In practice the plasmon angle is first measured by means of an ATR angle scan with a serum that does not contain the test antibody and then a second time after the antibody is added to the serum. From the shift in the two angular ATR scans the shift in plasmon angle is deduced. Therefore, measuring the shift in the angular position of the minimum in the reflectivity provides a quantitative-measure of the antibodies absorbed from the sample. A sensitive measurement of the antibodies absorbed is obtained because the measured ATR characteristics are strongly dependent upon the amount of antigens attached to the layer of antibodies. It should be noted that the primary physical quantity measured in this system as well as in most, if not all, current SPR sensors, is the total ATR beam intensity at each angle of incidence within a narrow angular interval, including the plasmon angle.
The theoretical basis for understanding the ATR phenomenon, associated with the resonant excitation of surface waves, is generally based on an infinite plane wave approximation for the incident light wave. While this method has been adequate to detect changes in surface properties, it would be beneficial to provide an improved method with greater sensitivity.
The above objectives as well as other objectives not specifically enumerated are achieved through the measurement of changes in optical properties of layered materials by first directing an incident wave toward layered materials under coupling conditions that will produce a propagating surface mode in the layered materials. Next, the intensity distribution within the transverse beam profile of the coupled output reflected beam is measured at a single value of the angle of incidence at or near the plasmon angle. Then, the index of refraction of the layered materials is modified and the intensity distribution within the transverse beam profile of the coupled output reflected beam is re-measured. Finally, the measured intensity distributions are compared to detect differences in the indices of refraction in the layered materials.
Another aspect of the present invention involves measuring changes in optical properties of layered materials by directing an incident wave toward layered materials under coupling conditions that will produce a propagating waveguide mode in the layered materials. Next, the intensity distribution within the transverse beam profile of the coupled output reflected beam is measured. Then, the index of refraction of the layered materials is modified and the intensity distribution within the transverse beam profile of the coupled output reflected beam is re-measured. Finally, the measured intensity distributions are compared to detect differences in the indices of refraction in the layered materials.
Various objects and advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments, when read in light of the accompanying drawings.